Method for evaluation of viability of viruses with lymphotropism properties

ABSTRACT

Methods and techniques to increase the reliability of detecting virus infections, particularly lymphotropism, to eliminate false negative reactions in testing blood for the presence of lymphotropic viruses during enzyme immunoassay (EIA) and polymerase chain reaction (PCR) testing, and to better detect viruses with lymphotropism in biological materials having a concentration of virus particles lower than the sensitivity threshold of existing EIA and PCR methods, thereby making the techniques of the present invention more reliable.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

The present invention is a continuation of U.S. patent application Ser.No. 16/799,087, filed Feb. 24, 2020, now U.S. Pat. No. ______, entitled“METHOD FOR EVALUATION OF VIABILITY OF VIRUSES WITH LYMPHOTROPISMPROPERTIES,” which is continuation of U.S. patent application Ser. No.15/966,513, filed Apr. 30, 2018, now U.S. Pat. No. 10,570,466, entitled“METHOD FOR EVALUATION OF VIABILITY OF VIRUSES WITH LYMPHOTROPISMPROPERTIES,” which is a continuation of U.S. patent application Ser. No.15/257,663, filed Sep. 6, 2016, now U.S. Pat. No. 9,957,579, entitled“METHOD FOR EVALUATION OF VIABILITY OF VIRUSES WITH LYMPHOTROPISMPROPERTIES,” which is a divisional of U.S. patent application Ser. No.14/397,680, filed Oct. 29, 2014, now U.S. Pat. No. 9,879,329, alsoentitled “METHOD FOR EVALUATION OF VIABILITY OF VIRUSES WITHLYMPHOTROPISM PROPERTIES,” which is a United States nationalization ofPCT/UZ2013/000001, filed May 21, 2013, and claims priority fromUzbekistanian Patent Application No. IAP 2012 0233, filed Jun. 18, 2012,entitled “METHOD FOR EVALUATION OF VIABILITY OF VIRUSES WITHLYMPHOTROPISM PROPERTIES,” the subject matters of which are incorporatedby reference herein in their entirety.

TECHNICAL FIELD

The present invention relates to methods of detection of viruses withlymphotropism properties in biological substrates with low concentrationof viral particles, evaluation of their viability and elimination offalse-negative results of enzyme immunoassay (EIA) and polymerase chainreaction (PCR) testing, and techniques related thereto that may be usedin the medical industry and biotechnology.

BACKGROUND OF THE INVENTION

The detection of viruses in biological substrates through isolation incell cultures is a well-known technique. As is known, viruses isolatedby cell culture methods are identified by haemadsorption,hemagglutination or indirect immunofluorescence methods. Proper samplingand short-time transportation to the laboratory venue on appropriatemedia are essential for effective isolation of viruses isolated inculture, which preserves virus viability and restricts bacteria andfungi reproduction. Many viruses, in particular the hepatitis B virus(HBV), the hepatitis C virus (HCV) and the human immunodeficiency virus(HIV), are anthroponotic viruses, i.e., affecting human cells only andthus causing diseases only to humans.

It should be understood that there are no experimental models of theseinfections. Also, there are no cultivated cell cultures, particularly inthe Republic of Uzbekistan, on which one may adequately studycytopathogenic properties and viability of these viruses in vitro.Moreover, because of the complexity, the isolation of viruses on cellcultures is not generally used for diagnostic purposes.

An immunological method for the detection of viruses in biologicalmaterial is known as an enzyme-linked, immunosorbent assay (ELISA),which is based on the use of specific viral proteins extracted frominfected cells or produced by genetic engineering, e.g., by thedetection and comparison of antibodies to the number of virus antigens.

In some virus infections, e.g., HCV, enzyme immunoassay (EIA) detectsthe antibodies only, thus substantially restricting evaluation of theprogress and activity of an infection. Moreover, EIA, in operation, hassensitivity threshold values, below which the detection of virusesbecomes impossible.

With regard to methods of detection of viruses with lymphotropismproperties in the biological materials, virus viability assessment, andthe exclusion of false-negative results of EIA and PCR, the closestanalog is the detection of viral RNA or DNA by the sampling ofbiological material and the detection of the presence of viral RNA orDNA by polymerase chain reaction (PCR).

The method and techniques of the instant invention relate to directmethods for the detection of the pathogen in biological materials,thereby permitting the evaluation of the activity of viral processes,where a positive PCR-reaction confirms the presence of the virus in theliver and in the blood with a high probability. However, PCRs of thebiological samples (plasma or blood proteins, tissue or organ biopsymaterials) do not always allow the detection of infections caused byviruses with lymphotropism properties, though such viruses may persistin substantially high concentrations in the lymphoid tissue(false-negative results of PCR), and vice versa a positive PCR may beobtained without persistence of viruses. Furthermore, PCR has asensitivity threshold, below which virus presence is not detectable bythis method, yet another drawback in the prior art. These and otherdrawbacks in the prior art lead to unreliable testing and detection ofviruses, particularly viruses with lymphotropism properties.

There is, therefore, a present need for an improved technique for thereliable detection of viruses, particularly those viruses withlymphotropism properties.

There is also a need for new techniques for the elimination of theaforementioned false-negative results when testing blood for theseviruses, particularly the elimination of false-negative results testingblood for the presence of lymphotropic viruses by EIA and PCR.

There is a further need for new techniques that can better detectviruses with lymphotropism properties in the biological materials wherevirus concentrations are low, for example, below the threshold ofcurrent IFA or PCR methods sensitivities.

These and many other objects are met in various embodiments of thepresent invention, offering significant advantages over the known priorart and consequent benefits to all mankind. The objects and features ofthe present invention, will become apparent in the detailed descriptionof the invention set forth below.

SUMMARY OF THE INVENTION

The present invention is directed to methods and techniques to increasethe reliability of detecting virus infections, particularlylymphotropism, to eliminate false negative reactions in testing bloodfor the presence of lymphotropic viruses during enzyme immunoassay (EIA)and polymerase chain reaction (PCR) testing, and to detect viruses withlymphotropism in biological material having a concentration of virusparticles lower than the sensitivity threshold of existing EIA and PCRmethods, thereby making the techniques of the present invention morereliable.

The methods and techniques of the present invention assess the viabilityof viruses with lymphotropism by collecting biological material anddetermining whether said material contains virus RNA or DNA, e.g., byconducting a polymerase chain reaction (PCR reaction). In addition, alymphocyte suspension is taken from the blood of healthy people, towhich lymphocytes an equal volume of biological material is added. Thiscombination is then mixed, preferably incubated at a temperature ofabout 37° C. for a period of about 6-8 hours, and the lymphocytes arewashed of plasma and broken down. The lymphocyte cytoplasm is thensubjected to PCR testing. The detection of virus RNA or DNA in thelymphocyte cytoplasm indicates that the viruses have retained theirviability. Correspondingly, the absence of virus RNA or DNA in thelymphocyte cytoplasm indicates the inactivation of the viruses. In thismanner the method and technique of the present invention allowsassessment of the viability of a variety of viruses, including the HBV,HCV and HIV viruses.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description is presented to enable any personskilled in the art to make and use the invention. For purposes ofexplanation, specific nomenclature is set forth to provide a thoroughunderstanding of the present invention. However, it will be apparent toone skilled in the art that these specific details are not required topractice the invention. Descriptions of specific applications areprovided only as representative examples. Various modifications to thepreferred embodiments will be readily apparent to one skilled in theart, and the general principles defined herein may be applied to otherembodiments and applications without departing from the scope of theinvention. The present invention is not intended to be limited to theembodiments shown, but is to be accorded the widest possible scopeconsistent with the principles and features disclosed herein.

The principles of the present invention are achieved through theevaluation of the viability of the viruses with lymphotropism propertiesby sampling of the biological material, detection of presence of viralRNA or DNA using the polymerase chain reaction (PCR), as brieflydiscussed hereinabove. As described, a lymphocytes suspension isobtained from healthy human blood and an equal amount of biologicalmaterial is then added. The aforesaid admixture is stirred, and thenincubated at about 37° C. for about 6-8 hours, resulting in thewashing-out of the lymphocytes from the plasma, and the lymphocytesbeing destroyed. The lymphocytes' cytoplasm is then subjected to a PCRtest.

As discussed, the detection of viral RNA or DNA in the cytoplasm oflymphocytes indicates the preserved viability of viruses, whereas theabsence of viral RNA or DNA viruses in the cytoplasm of lymphocytesindicates inactivation of viruses. It should be understood that plasmaor blood serums, biopsy samples of tissue or organs, and the washoutsfrom the medical instruments may be used as the biological samples. Inthis manner, the methodologies and techniques of the instant inventionallow assessment of the viability of viruses, particularly the HBV, HCVand HIV viruses.

As discussed, another objective of the present invention is theelimination of EIA and PCR false-negative results, such as by obtainingblood from patients suspected of being infected by lymphotropic viruses.For this approach, about 6-8 ml of such blood is drawn into test tubes,which preferably contain about 2.0 ml normal saline and about 2-3 dropsof heparin. The lymphocytes are then separated from the blood andincubated at about 37° C. for about 6-8 hours, where lymphocytes arewashed-out from the plasma and destroyed, and the cytoplasms oflymphocytes are then subjected to PCR. As discussed hereinabove, thedetection of viral RNA or DNA in the lymphocytes' cytoplasm indicatesthe presence of viruses, whereas the absence of viral RNA or DNA in thelymphocytes' cytoplasm indicates the absence of viruses in the blood.The content of a patient's lymphocyte is thus subjected to PCR-testing.

It is known that many viruses, particularly those of the aforementionedhepatitis B virus (HBV), hepatitis C virus (HCV) and humanimmunodeficiency virus (HIV) can replicate in mononuclear blood cells,particularly, in the lymphocytes and in macrophages. It is known thatHBV- and HCV-infections simultaneously cause inflammatory processes inthe liver with subsequent hepatitis, as well as secondaryimmunodeficiency with various degrees of T- lymphopenia andB-lymphopenia, imbalance of regulatory subpopulations of T-lymphocytes(T-helpers and T-suppressors), reduction of immune regulatory index(IRI), and dysgammaglobulinemia. The degree and grade ofimmunodeficiency, however, has been found to have no relation to thedegree of the pathologic process in the liver. Indeed, patients withchronic HBV and HCV infections have different intensities ofpathological processes in the liver tissue after some time, from weak toexpressed, but nonetheless have stable and steady aggravation ofsecondary immunodeficiencies.

The dissociation of the degree of liver tissue injury and the degree ofsecondary immunodeficiency in various nosological forms of chronic virushepatitis supports the idea that the hepatitis and secondaryimmunodeficiency in HBV and HCV infections are associated, mutuallyaggravating, but not mutually conditional. In other words, HBV and HCV,along with hepatotropic property viruses, possess expressed lymphotropicproperties—direct properties that cause secondary immunodeficiencies.The differences in clinical appearances of the liver tissue injuries andthe degree of immunodeficiency in HBV and HCV infections are due todifferences in the degree of hepatotropic and lymphotropic properties ofthese viruses. Thus, the differences in the degrees of hepatotropic andlymphotropic properties of viruses determine the differences of thepathogenesis, clinical appearance and the pattern of antiviral therapyeffects in chronic HBV and HCV infections in various stages of thesediseases.

The identity of the lymphotropic properties of the aforesaid HBV andHCV, and HIV viruses, besides secondary immunodeficiency forming, isconfirmed also by commonality of their epidemiological features,mechanism of transfer, progress of associated opportunistic infections(frequent respiratory diseases, intestinal infections), and particularlythe development of the lymphogranulomatosis in different tissues of theorganism. The development of lymphoid follicles' clusters, which is theaforementioned lymphogranulomatosis, in various organs and tissues ofthe organism is considered intrinsic for viral infections of thelymphoid cell system.

When considering the lymphotropic properties of HBV, it was found thatregardless of the serum titer, HBV can permanently persist in highconcentrations in the cytoplasm of lymphoid elements. This phenomenon isused in the context of the instant invention for reliable increasing andelimination of the aforesaid false-negative results by EIA and PCR, andthe detection of lymphotropic viruses in biological material withconcentrations of viral particles below the threshold oftest-sensitivity for the EIA and PCR techniques, as generally describedhereinabove.

In the instant invention, Applicant employed the lymphotropic propertiesof HBV, HCV and HIV in an evaluation method of virus viability—theability of these viruses to penetrate and persist intracellularly in thehealthy human lymphocytes during their in vitro incubation.

It should be understood that the evaluation of the viability of viruseswith lymphotropism properties, particularly HBV, HCV and HIV, requiredthe long-term storage of viruses, and the control of the antiviralefficiency of various disinfecting chemicals and physical factorsagainst these viruses, as well as the control of antiviral therapy, asdescribed in more detail hereinbelow.

Below is a description of a method pursuant to the teachings of anembodiment of the present invention directed to the evaluation of theviability of viruses with lymphotropism properties.

I. Producing a Suspension of Viruses with Lymphotropism Properties.

In the production of a suspension of viruses, the biological material(plasma or blood serum, biopsy samples of tissue or organs, and/or thewash-outs from medical instruments) is obtained. Then the biologicalmaterial is subjected to quantitative PCR for the verification of thepresence of viruses with lymphotropism properties, and thequantification of titer of the viruses. Viruses contained in thebiological material are kept in a frozen state in a refrigerator atbelow about −25° C. temperature.

II. Producing a Lymphocytes Suspension from a Healthy Human Subject.

a) healthy volunteers are tested for infection with lymphotropic virusesusing EIA, as described. Lymphocytes from healthy people with a negativeresult for study viruses are used in the investigations;

b) to receive a sufficient amount of lymphocyte, blood is taken from anulnar vein in an amount of about 20-30 ml in the morning from a fasting,healthy human subject. Then about 7-8 ml of the blood is transferred torespective centrifuge tubes containing about 2 ml normal saline andabout 3 drops of heparin (“Heparin” concentration of 5000 ME/ml; 3 dropscontain 750 ME/ml of heparin). The resulting solution is then stirredthoroughly;

c) the lymphocytes are separated from the whole heparin containing bloodin a ficoll-verografin gradient with density d=1.077 g/ml pursuant to atechnique known in the art. Then, about 2 ml of the aforesaidficoll-verografin gradient is poured into a clean centrifuge tube, thenthe heparinized blood lays on its surface and the tube is centrifuged atabout 1500 RPM for about 20 minutes. During centrifugation, all bloodcells, excluding lymphocytes, penetrate through the aforesaidficoll-verografin gradient. Blood plasma, however, remains above thegradient. In the border of the ficoll-verografin gradient and theplasma, there is a peculiar turbid ring, consisting of pure lymphocytesso formed. The ring with lymphocytes is then carefully pumped with apipette and transferred to a clean centrifugal tube;

d) the lymphocytes are then washed-out with about 10 ml of normal saline2-3 times with further centrifugation at about 1500 RPM for about 20minutes; and

e) after the last centrifugation in the previous step, the supernatantis removed. The sediment containing lymphocytes is diluted andre-suspended in about 600 μl of normal saline. It should be understoodthat a lymphocytes suspension so produced may be stored no more thanabout a day at a temperature of about +4° C.

III. Evaluation of Viruses with Lymphotropism Properties to Penetrateand Persist Intracellulary in the Human Lymphocytes In Vitro.

1) Biological material containing viruses with lymphotropism propertiesis taken from a refrigerator and thawed at room temperature;

2) an equal amount (about 300 μl) of virus-containing biologicalmaterial and a suspension of healthy human lymphocytes is transferredwith a pipette to a clean centrifugal tube, and the contents are mixedand placed for incubation (incubation of viruses with lymphocytes invitro) into a thermostat at about +37° C. for about 6-8 hours. Thetesting tube is preferably mixed with shacking every 1.5-2 hours;

3) The washing-out of lymphocytes is then done. The aforesaid testingtube is removed from the thermostat. About 6-8 ml of normal saline isadded, mixed and the admixture centrifuged at about 1500 RPM for about20 minutes. The lymphocytes are then sediment at the bottom of the tube.The supernatant (mixture of plasma with saline) is then entirelyremoved.

The lymphocytes are washed out in normal saline and sediment 2-3 times.After the last centrifugation and supernatant removal, the suspension ofthe lymphocytes (sediment) is diluted with about 500 μl of normal salineand transferred to a plastic 1.5 lock tube (Eppendorf tube) or similartube;

4) Thereafter, the tube is placed into a freezer, such as a house graderefrigerator, overnight. The lymphocytes are thereby destroyed underthese slow freezing conditions;

5) The removal of the membrane of destroyed lymphocytes. The next daythe tubes from the freezer are thawed at room temperature. Then themembranes of the aforesaid destroyed lymphocytes are removed bycentrifugation at about 3000 RPM for about 30 minutes. Membranes areprecipitated on the bottom of the tube and the lymphocytes' cytoplasmcontent remains in the supernatant; and

6) The supernatant from the tube is transferred and subjected toquantitative PCR for testing for possible viral RNA or DNA in thecytoplasm of the lymphocytes that were previously in the infectedpatient's plasma.

IV. Assessment of the Results.

1. Positive PCR for the presence of viral RNA or DNA in the cytoplasm oflymphocytes indicates the remaining virus viability, i.e., the virus'ability to penetrate and persist in human lymphocytes in vitro.

2. Negative PCR for the presence of viral RNA or DNA in the cytoplasm oflymphocytes indicates the loss (inactivation) of virus viability, i.e.,the loss of the virus' ability to penetrate and persist in humanlymphocytes in vitro.

The following examples further support the presently-claimed invention:

EXAMPLE #1: THE ASSESSMENT OF VIABILITY OF VIRUSES WITH LYMPHOTROPISMPROPERTIES

The blood is obtained from an ulnar vein of a patient after receivingantiviral therapy for hepatitis C. The plasma is then separated fromwhole blood and subjected to quantitative PCR for the verification ofthe presence of HCV and quantification virus titer. In this embodiment,the PCR testing is negative. Tested plasma is kept in the freezer atbelow −25° C. temperature.

Simultaneously a 20-30 ml sample of blood from healthy human volunteersis obtained in the morning from an ulnar vein. The blood plasma portionis then subjected to PCR analysis for viruses with lymphotropismproperties infection. The lymphocytes from the healthy humans, withnegative testing results for infection, are used for furtherinvestigation. Then 7-8 ml blood aliquots are transferred to centrifugetubes containing about 2 ml of normal saline and about 3 heparin drops(“Heparin” concentration is 5000 ME/ml, 3 drops contain 750 ME/ml ofheparin). The solution in the tube is then mixed thoroughly. Asdescribed hereinabove, the lymphocytes are separated from the wholeheparinized blood in a ficoll-verografin gradient with d=1.077 g/mldensity according to a known method by Garib, Yu et al.

Then, about 2 ml of the aforesaid ficoll-verografin gradient is pouredinto a clean centrifuge tube, where heparinized blood lays on thesurface of the gradient and then is centrifuged at about 1500 RPM forabout 20 minutes.

All blood cells, excluding lymphocytes, penetrate the ficoll-verografingradient and the sediment underneath. The blood plasma is found abovethe aforesaid gradient. Along the border between the ficoll-verografingradient and the plasma, the afore-noted peculiar turbid ring with purelymphocytes suspension is formed. The ring with lymphocytes is thencarefully sucked up with a pipette, and transferred to a cleancentrifuge tube. The lymphocytes are washed out in normal saline andsedimented about 2-3 times. After the last centrifugation, thesupernatant is removed. The sediment containing lymphocytes is thendiluted with about 600 μl saline and re-suspended. As is understood, thelymphocytes suspension so formed may be stored for about 1 day at about+4° C. temperature.

The testing plasma from the freezer is then thawed at room temperature.Equal volumes (about 300 μl) of plasma and the suspension of lymphocytesare transferred to a clean centrifuge tube with a pipette, mixed andplaced for incubation in a thermostat at about +37° C. temperature forabout 6-8 hours. The tube is then mixed by shaking every about 1.5-2hours.

After incubation, the tube is removed from the thermostat. Then, about6-8 ml of saline is added, mixed and centrifuged at about 1500 RPM forabout 20 minutes. As discussed, the lymphocytes sediment at the bottomof the tube. The supernatant (mixture of plasma and normal saline) isthen removed. With 2-3 times wash-out in normal saline and thelymphocytes sedimentation is performed in the same fashion. After thelast centrifugation, the supernatant is removed, and a suspension oflymphocytes (sediment) is diluted by adding about 300 μl of normalsaline, and transferred to a 1.5 ml lock tube, such as an Eppendorftube.

Thereafter, lymphocyte membranes are destroyed by placing them overnightin a house-grade freezer. On the next day, the tubes are thawed at roomtemperature. Then, the membranes of the destroyed lymphocytes areremoved from the suspension by centrifugation of the tube at about 3000RPM for about 30 minutes. The membranes are thereby precipitated at thebottom of the tubes, and the lymphocyte cytoplasm contents remain in thesupernatant, as also described hereinabove. The supernatant is thentransferred from the tube and subjected to a quantitative PCR test forthe presence of HCV viruses in the cytoplasm of the lymphocytes. Apositive PCR test for HCV, of course, indicates the preservation of theHCM viability and the requirement of further antiviral therapy.

EXAMPLE #2

A liver tissue, such as sampled by a liver puncture of a patient, whowas given antiviral therapy for hepatitis B is obtained. A liver biopsysample thereof is homogenized in an about 1.5 ml normal saline;transferred to a centrifuge tube, and then centrifuged at about 1500 RPMfor about 20 minutes; and the supernatant transferred to a tube. Onepart of the supernatant is then subjected to quantitative PCR testingfor the presence of HCV virus and quantification of virus titer. If thePCR test for HCV is positive, the biopsy sample is kept in the freezerin the refrigerator at below −25° C. temperature.

A lymphocyte suspension from a healthy human is made, as describedhereinabove in connection with Example #1. The supernatant from theaforesaid liver biopsy sample homogenate is thawed at room temperature.Then equal volumes (about 300 μl) of the supernatant and lymphocytesuspension are added to a tube by an automatic pipette or similar suchmeans; the resulting solution is admixed and placed for incubation intoa thermostat at about +37° C. temperature for about 6-8 hours, where thetesting tube is mixed by shaking about every 1.5-2 hours.

The tube is the removed from thermostat and about 6-8 ml of normalsaline is added, admixed and centrifuged at about 1500 RPM for about 20minutes. The lymphocytes sediment at the bottom of the tube, asdescribed hereinabove.

The supernatant (mixture of plasma with normal saline) is removedentirely, and treated by a 2-3 times wash-out in normal saline, wherethe aforementioned lymphocytes sedimentation is performed in the samefashion as before. After the last centrifugation, the supernatant isremoved and the suspension of lymphocytes (in the sediment) is dilutedby adding about 300 μl of normal saline. Thereafter, the destruction ofthe lymphocyte membranes is performed by putting the testing tube into ahouse-grade freezer overnight.

Accordingly, lymphocyte membranes are destroyed by overnight placementinto house-grade freezer. On the next day, the tubes are thawed at roomtemperature. Then, the membranes of destroyed lymphocytes are removedfrom suspension by centrifugation of the tube at about 3000 RPM forabout 30 minutes. The membranes are precipitated along the bottom of thetubes and the lymphocyte cytoplasm contents remain in the supernatant,as discussed and described hereinabove. The supernatant is transferredfrom the tube and subjected to a quantitative PCR test for the presenceof HBV virus in the cytoplasm of the lymphocytes, where a negative PCRtest for HBV indicates the virus' loss of viability (inactivation).

EXAMPLE #3

This example concerns the detection of viruses with lymphotropismproperties in biological material with the concentration of virus belowEIA and PCR sensitivity thresholds.

In a blood center, the blood plasma from about 6-8 ml of blood is testedfor viruses with lymphotropism properties. One part of the plasma issubjected to quantitative PCR testing for the presence of HBV, HCV orHIV viruses, and the quantification of virus titer, where the PCR testhere for the presence of viruses is negative. The tested plasma isstored in a freezer at below about −25° C.

The lymphocyte suspension from healthy human subjects is then preparedas described in more detail hereinabove in connection with Example #1.

The testing plasma from the freezer is thawed at room temperature. Equalvolumes (about 300 μl) of plasma and the suspension of lymphocytes istransferred to a clean centrifuge tube using an automatic pipette,admixed and placed for incubation in a thermostat at about +37° C. forabout 6-8 hours. The tube is mixed by shaking about every 1.5-2 hours.

The tube is then removed from the thermostat, and about 6-8 ml of normalsaline is added, admixed and centrifuged at about 1500 RPM for about 20minutes. As described hereinabove, the lymphocytes sediment along thebottom of the tube. The supernatant (mixture of plasma with normalsaline) is removed entirely, and the remainder 2-3 times wash-out innormal saline, where the lymphocytes sedimentation is performed in thesame manner as set forth hereinabove. After the last centrifugation, thesupernatant is removed and the suspension of lymphocytes (sediment) isdiluted by adding about 300 μl of normal saline.

Thereafter, the lymphocyte membranes are destroyed by placing them intoa house-grade freezer overnight. On the next day, the tubes are thawedat room temperature. The membranes of the destroyed lymphocytes areremoved from suspension by centrifugation of the tube at about 3000 RPMfor about 30 minutes. As discussed, the membranes precipitated along thebottom of the tubes, and the lymphocyte cytoplasm contents remain in thesupernatant. The supernatant is then transferred from the tube andsubjected to quantitative PCR testing for the detection of HBV, HCV andHIV viruses in the content of lymphocytes' cytoplasm, where a positivePCR for HCV indicates the presence of HCV virus in the donor plasma,indicating that donor's ineligibility for transfusion.

EXAMPLE #4: THE ELIMINATION OF EIA AND PCR FALSE-NEGATIVE RESULTS

In this example, about 6-8 ml of blood is obtained in the morning from afasting donor, preferably from an ulnar vein. Whole blood is thentransferred to a tube, subjected to sedimentation techniques, asdescribed herein, and a serum is obtained; one part of the serum issubjected to a PCR test for the presence of HBV, HCV or HIV viruses. andthe quantification of virus titer, where the PCR tests are negative. Therest of the blood serum is stored in the tube. The lymphocyte suspensionfrom a healthy human subject is performed, as described in more detailhereinabove in connection with Example #1.

The lymphocytes are separated and destroyed by overnight freezing in ahouse-grade refrigerator, as described. On the next day, the tube isthawed at room temperature. Then the membranes of the destroyedlymphocytes are removed from the suspension by centrifugation at about3000 RPM for about 30 minutes. The membranes precipitate on the bottomof the tube, and lymphocyte cytoplasm contents remain in thesupernatant. The supernatant is then transferred from the tube andsubjected to a quantitative PCR test for the detection of HBV, HCV andHIV viruses in the cytoplasm of the lymphocytes, where a positive PCRfor HBV indicates the presence of HBV in the donor blood.

In a standard PCR test, the detection rate of HBV and HCV has beenobserved at about 2.7%. According to epidemiological data, new cases ofhepatitis B (HBV) and hepatitis C (HCV) transfer occur due to thetransfusion of infected blood or its components in about 2.2%-5.6% ofoccurrences. To uncover the reasons behind this and techniques for theelimination of HBV infection in recipients, the lymphotropic propertiesof the virus were used.

In particular, serums from 309 donor blood samples were tested by PCRfor HBV markers detection rate.

PCR revealed HBV in 6 out of 209 serum samples that estimated at about1.94% of all number of donors' sample. The same PCR study (study oflymphocytes content from the same donors) revealed HBV in 17 out of 309samples, estimated at about 7.44% of all donors' samples.

Thus, the standard PCR testing of blood serum was false-negative in5.50% of samples, which indicates that this is the reason for HBVinfection in recipients by transfusion of infected blood or thecomponents thereof.

While the present invention has been illustrated by the description ofthe embodiments thereof, and while the embodiments have been describedin detail, it is not the intention of the Applicant to restrict or inany way limit the scope of the appended claims to such detail.Additional advantages and modifications will readily appear to thoseskilled in the art. Therefore, the invention in its broader aspects isnot limited to the specific details, representative apparatus andmethod, and illustrative examples shown and described. Accordingly,departures may be made from such details without departure from thebreadth or scope of the applicant's concept. Furthermore, although thepresent invention has been described in connection with a number ofexemplary embodiments and implementations, the present invention is notso limited but rather covers various modifications and equivalentarrangements, which fall within the purview of the appended claims.

What is claimed is:
 1. A method for the detection of viruses withlymphotropism properties in biological material, comprising: collectinga biological material from a patient, wherein said biological materialhas a low virus concentration therein; forming an admixture of an equalamount of said biological material from said patient with a lymphocytesuspension from a healthy human subject; stirring and incubating theadmixture; washing-out the lymphocytes in the admixture; concentratinglymphocyte cytoplasm content within said admixture by removinglymphocyte membranes from the admixture; and detecting, within saidlymphocyte cytoplasm content, the presence or absence of the virus RNAor DNA, wherein the presence of the virus RNA or DNA in the lymphocytecytoplasm content, with an earlier non-detection of infection, indicatesthe preserved viability of viruses in said biological sample of saidpatient, wherein the absence of the virus RNA or in the lymphocytecytoplasm content indicates the loss of virus viability in saidbiological sample of said patient.
 2. The method according to claim 1,wherein said low virus concentration is below enzyme immunoassay (EIA)and polymerase chain reaction (PCR) testing sensitivity thresholds. 3.The method according to claim 1, wherein said biological materials areselected from the group consisting of plasma, blood serum, biopsytissues or organs, medical instrument washouts, and combinationsthereof.
 4. The method according to claim 1, wherein the virusesdetected are selected from the group consisting of HBV, HCV, HIV, andcombinations thereof.
 5. The method according to claim 1, wherein saidadmixture is incubated at about 37° C. for about 6-8 hours.
 6. Themethod according to claim 1, wherein the detecting of the virus RNA orDNA in the lymphocyte cytoplasm content is done by polymerase chainreaction (PCR).
 7. The method according to claim 1, wherein, in saidstep of forming, said lymphocyte suspension is from a blood sample ofsaid healthy human subject.
 8. The method according to claim 1, wherein,prior to said step of incubating, the admixture is stirred.
 9. Themethod according to claim 1, wherein, in said step of removinglymphocyte membranes, the lymphocyte membranes are separated bycentrifugation.
 10. The method according to claim 1, wherein theviability of said virus RNA or DNA is the ability of the virus RNA orDNA to penetrate and persist intracellularly in the lymphocytes of atleast one healthy human subject during in vitro incubation.
 11. Themethod according to claim 1, wherein, in said step of removing,lymphocytes within said admixture are destroyed.
 12. The methodaccording to claim 10, wherein the lymphocytes are destroyed byfreezing.
 13. The method according to claim 1, wherein, in said step ofwashing-out, the lymphocytes are washed out from the plasma portion inthe admixture.
 14. A method for the detection of viruses withlymphotropism properties in biological material, comprising: collectinga biological material from a patient, wherein said biological materialhas a low virus concentration; collecting a lymphocyte suspension from ablood sample of at least one healthy human subject; forming an admixtureof an equal amount of said biological material with said lymphocytesuspension; stirring and incubating the admixture; washing-out thelymphocytes from the admixture; concentrating lymphocyte cytoplasmcontent by removing lymphocyte membranes from the admixture, separatingthe lymphocyte cytoplasm content by centrifugation; and detecting,within the lymphocyte cytoplasm content, the presence or absence of thevirus RNA or DNA, wherein the presence of the virus RNA or DNA in thelymphocyte cytoplasm content, with an earlier non-detection ofinfection, indicates the preserved viability of viruses in saidbiological sample of said patient, wherein the absence of the virus RNAor in the lymphocyte cytoplasm content indicates the loss of virusviability in said biological sample of said patient.
 15. The methodaccording to claim 14, wherein said low virus concentration is belowenzyme immunoassay (EIA) and polymerase chain reaction (PCR) testingsensitivity thresholds.
 16. The method according to claim 14, wherein,in said step of washing-out, the lymphocytes are washed out from theplasma portion in the admixture.
 17. The method according to claim 14,wherein said biological materials are selected from the group consistingof plasma, blood serum, biopsy tissues or organs, medical instrumentwashouts, and combinations thereof.
 18. The method according to claim14, wherein the viruses detected are selected from the group consistingof HBV, HCV, HIV, and combinations thereof.
 19. The method according toclaim 14, further comprising, after the step of forming said admixture,the step of: incubating said admixture at about 37° C. for about 6-8hours.
 20. The method according to claim 19, wherein, prior to said stepof incubating, the admixture is stirred.
 21. The method according toclaim 14, wherein the detecting of the virus RNA or DNA in thelymphocyte cytoplasm content is done by polymerase chain reaction (PCR).22. The method according to claim 14, wherein, in said step of forming,said lymphocyte suspension is from a blood sample of said at least onehealthy human subject.